Felicia Grasso
Dec 1, 2020; 19:1986-1996
Research

Tricia Rowlison
Dec 1, 2020; 19:2090-2103
Research

Simone Kurz
Dec 29, 2020; 0:RA120.002266v1-mcp.RA120.002266
Research

Kyle Swovick
Dec 28, 2020; 0:RA120.002301v1-mcp.RA120.002301
Research

Weixian Deng
Dec 22, 2020; 0:RA120.002411v1-mcp.RA120.002411
Research

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
Research Articles

Melissa Gómez
Dec 1, 2020; 61:1658-1674
Research Articles

Genta Kakiyama
Dec 1, 2020; 61:1629-1644
Research Articles

In SAS Customer Intelligence Studio, you might notice that the user interface becomes unresponsive, as shown below: imgalt="SAS Customer Intelligence Studio UI becomes unresponsive" src="{fusion_66537

In SAS Customer Intelligence Studio, you can choose to create a new calculated variable in the Edit Value dialog box when you populate a treatment custom detail. Following creation of the new calculated

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Functions of the gut microbiome have a growing number of implications for host metabolic health, with diet being one of the most significant influences on microbiome composition. Compelling links between diet and the gut microbiome suggest key roles for various macronutrients, including lipids, yet how individual classes of dietary lipids interact with the microbiome remains largely unknown. Sphingolipids are bioactive components of most foods and are also produced by prominent gut microbes. This makes sphingolipids intriguing candidates for shaping diet–microbiome interactions. Here, we used a click chemistry–based approach to track the incorporation of bioorthogonal dietary omega-alkynyl sphinganine (sphinganine alkyne [SAA]) into the murine gut microbial community (Bioorthogonal labeling). We identified microbial and SAA-specific metabolic products through fluorescence-based sorting of SAA-containing microbes (Sort), 16S rRNA gene sequencing to identify the sphingolipid-interacting microbes (Seq), and comparative metabolomics to identify products of SAA assimilation by the microbiome (Spec). Together, this approach, termed Bioorthogonal labeling-Sort-Seq-Spec (BOSSS), revealed that SAA assimilation is nearly exclusively performed by gut Bacteroides, indicating that sphingolipid-producing bacteria play a major role in processing dietary sphinganine. Comparative metabolomics of cecal microbiota from SAA-treated mice revealed conversion of SAA to a suite of dihydroceramides, consistent with metabolic activities of Bacteroides and Bifidobacterium. Additionally, other sphingolipid-interacting microbes were identified with a focus on an uncharacterized ability of Bacteroides and Bifidobacterium to metabolize dietary sphingolipids. We conclude that BOSSS provides a platform to study the flux of virtually any alkyne-labeled metabolite in diet–microbiome interactions.

The cornea is densely innervated, mainly by sensory nerves of the ophthalmic branch of the trigeminal ganglia (TG). These nerves  are important to maintain corneal homeostasis, and nerve damage can lead to a decrease in wound healing, an increase in corneal ulceration and dry eye disease (DED), and neuropathic pain. Pathologies, such as diabetes, aging, viral and bacterial infection, as well as  prolonged use of contact lenses and surgeries to correct vision can produce nerve damage. There are no effective therapies to alleviate DED (a multifunctional disease) and several clinical trials using -3 supplementation show unclear and sometimes negative results. Using animal models of corneal nerve damage, we show that treating corneas with pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) increases nerve regeneration, wound healing, and tear secretion. The mechanism involves the activation of a calcium-independent phospholipase A2 (iPLA2) that releases the incorporated DHA from phospholipids and enhances the synthesis of docosanoids neuroprotectin D1 (NPD1) and a new resolvin stereoisomer  RvD6i. NPD1 stimulates the synthesis of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and of semaphorin 7A (Sema7A).  RvD6i treatment of injured corneas modulates gene expression in the TG resulting in enhanced neurogenesis; decreased neuropathic pain and increased sensitivity. Taken together, these results represent a promising therapeutic option to re-establish the homeostasis of the cornea.

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human iPS cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in C. reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms (CRPs) in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that CRPs mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function. 

TG-rich lipoprotein (TRL)-related biomarkers, including TRL-cholesterol (TRL-C), remnant-like lipoprotein particle-cholesterol (RLP-C), and apoC-III have been associated with atherosclerosis. However, their prognostic values have not been fully determined, especially in patients with previous CAD. This study aimed to examine the associations of TRL-C, RLP-C, and apoC-III with incident cardiovascular events (CVEs) in the setting of secondary prevention of CAD. Plasma TRL-C, RLP-C, and total apoC-III were directly measured. A total of 4,355 participants with angiographically confirmed CAD were followed up for the occurrence of CVEs. During a median follow-up period of 5.1 years (interquartile range: 3.9–6.4 years), 543 (12.5%) events occurred. Patients with incident CVEs had significantly higher levels of TRL-C, RLP-C, and apoC-III than those without events. Multivariable Cox analysis indicated that a log unit increase in TRL-C, RLP-C, and apoC-III increased the risk of CVEs by 49% (95% CI: 1.16–1.93), 21% (95% CI: 1.09–1.35), and 40% (95% CI: 1.11–1.77), respectively. High TRL-C, RLP-C, and apoC-III were also independent predictors of CVEs in individuals with LDL-C levels ≤1.8 mmol/l (n = 1,068). The addition of RLP-C level to a prediction model resulted in a significant increase in discrimination, and all three TRL biomarkers improved risk reclassification. Thus, TRL-C, RLP-C, and apoC-III levels were independently associated with incident CVEs in Chinese CAD patients undergoing statin therapy.

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter allopregnanolone (AlloP) levels, an important neurosteroid in the brain. We assessed the levels and regulation of AlloP and the effects of AlloP supplementation on cognitive function in 4-month-old and 24-month-old male C57BL/6 mice. With age, the expression of enzymes involved in the AlloP synthetic pathway was decreased and corticosterone (CORT) synthesis increased. Supplementation of AlloP improved cognitive function. Interestingly, interleukin 6 (IL-6) infusion in young animals significantly reduced the production of AlloP compared with controls. It is notable that inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein 130, significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of AlloP and increases CORT levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in AlloP levels and an increase in CORT. Furthermore, our results indicate that AlloP is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR, were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1^WT versus Kir2.1^314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

Natural compounds that can stimulate salivary secretion are of interest in developing treatments for xerostomia, the perception of a dry mouth, that affects between 10 and 30% of the adult and elderly population. Chemesthetic transient receptor potential (TRP) channels are expressed in the surface of the oral mucosa. The TRPV1 agonists capsaicin and piperine have been shown to increase salivary flow when introduced into the oral cavity but the sialogogic properties of other TRP channel agonists have not been investigated. In this study we have determined the influence of different TRP channel agonists on the flow and protein composition of saliva. Mouth rinsing with the TRPV1 agonist nonivamide or menthol, a TRPM8 agonist, increased whole mouth saliva (WMS) flow and total protein secretion compared with unstimulated saliva, the vehicle control mouth rinse or cinnamaldehyde, a TRPA1 agonist. Nonivamide also increased the flow of labial minor gland saliva but parotid saliva flow rate was not increased. The influence of TRP channel agonists on the composition and function of the salivary proteome was investigated using a multi-batch quantitative MS method novel to salivary proteomics. Inter-personal and inter-mouth rinse variation was observed in the secreted proteomes and, using a novel bioinformatics method, inter-day variation was identified with some of the mouth rinses. Significant changes in specific salivary proteins were identified after all mouth rinses. In the case of nonivamide, these changes were attributed to functional shifts in the WMS secreted, primarily the over representation of salivary and nonsalivary cystatins which was confirmed by immunoassay. This study provides new evidence of the impact of TRP channel agonists on the salivary proteome and the stimulation of salivary secretion by a TRPM8 channel agonist, which suggests that TRP channel agonists are potential candidates for developing treatments for sufferers of xerostomia.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

Extracellular vesicles (EVs) secreted by the epididymal epithelium transfer to spermatozoa key proteins that are essential in promoting motility and subsequent fertilization success. Using the domestic cat model, the objectives were to (1) characterize and compare protein content of EVs between segments of the epididymis, and (2) compare EV protein compositions between normo- and teratospermic individuals (producing >60% of abnormal spermatozoa). Epididymal EVs from adult cats were isolated and assessed via liquid chromatography tandem MS. Both male types shared 3008 proteins in total, with 98 and 20 EV proteins unique to normospermic and teratospermic males, respectively. Expression levels of several proteins changed between epididymal segments in both male types. Several proteins in both groups were related to sperm motility (e.g. hexokinase 1, adenylate kinase isoenzyme) and zona pellucida or oolemma binding (e.g. disintegrin and metalloproteinase domain proteins, zona binding proteins 1 and 2). Interestingly, seven cauda-derived EV proteins trended downward in teratospermic compared with normospermic males, which may relate to poor sperm quality. Collective results revealed, for the first time, EV proteins related to sequential sperm maturation with differences observed between normospermic and teratospermic individuals.

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles. In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N-termini, and then the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1,550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N-termini registered in the Swiss-Prot database. Since this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N-termini, including proteoforms with neo-N-termini.

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of co-eluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias towards high abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or Carboxylate modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid-phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH RP. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low abundance bait proteins.

Cells continually degrade and replace damaged proteins. However, the high energetic demand of protein turnover generates reactive oxygen species (ROS) that compromise the long-term health of the proteome. Thus, the relationship between aging, protein turnover and energetic demand remains unclear. Here, we used a proteomic approach to measure rates of protein turnover within primary fibroblasts isolated from a number of species with diverse lifespans including the longest-lived mammal, the bowhead whale. We show that organismal lifespan is negatively correlated with turnover rates of highly abundant proteins. In comparison to mice, cells from long-lived naked mole rats have slower rates of protein turnover, lower levels of ATP production and reduced ROS levels. Despite having slower rates of protein turnover, naked mole rat cells tolerate protein misfolding stress more effectively than mouse cells. We suggest that in lieu of rapid constitutive turnover, long-lived species may have evolved more energetically efficient mechanisms for selective detection and clearance of damaged proteins.

High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.

Russia’s aggression and a crisis for multilateralism 30 March 2023 — 1:00PM TO 2:00PM Anonymous (not verified) 23 March 2023 Chatham House and Online

In conversation with Dmytro Kuleba, minister of foreign affairs of Ukraine, about how multilateral organizations struggle to respond adequately to Russia’s full-scale invasion of Ukraine.

As a permanent member of the UN Security Council, Russia pursues this war in defiance of the umbrella organization’s multiple resolutions condemning the invasion, along with its war crimes, annexation of territory, deliberate targeting of civilian nuclear infrastructure, cultural extermination, and global disinformation campaign. 

This event explores the following questions: 

• How should the response of the UN to Russian aggression be assessed? 
• What can be done to uphold the guiding principles of the UN Charter? 
• Is there a ‘UN problem’ or a ‘Russia problem’? 
• How can trust and the legitimacy of multilateral cooperation be restored in times of strategic rivalry and rising global tensions?
• Who can drive such an effort? 
• Is post-Soviet transfer of the UN Security Council seat to Russia a cause of current impunity? 

This event features a live in-person audience in Kyiv as well as in London and online.

Australian Prime Minister Anthony Albanese said Thursday his government will introduce legislation to ban children under 16 years of age from social media.

MLCommons this week issued the results of its latest MLPerf Inference (v3.1) benchmark exercise. Nvidia was again the top performing accelerator, but Intel (Xeon CPU) and Habana (Gaudi1 and 2) […]

The post MLPerf Releases Latest Inference Results and New Storage Benchmark appeared first on HPCwire.

Microsoft and Quantinuum reported the ability to create 12 logical qubits on Quantinuum’s H2 trapped ion system this week and also reported using two logical qubits on an H1 system […]

The post Microsoft, Quantinuum Use Hybrid Workflow to Simulate Catalyst appeared first on HPCwire.

BUFFALO, N.Y., Nov. 11, 2024 — Motivair Corporation, a leading global provider of advanced liquid cooling solutions, will be showcasing its cutting-edge MI300X Dynamic Cold Plates at the upcoming International […]

The post Motivair to Showcase MI300X Cold Plates at SC24 for AI Servers appeared first on HPCwire.

SpaceX has scrubbed Tuesday's launch of 20 Starlink Internet satellites into orbit from the Vandenberg Space Force Base in California until Wednesday, adding to its massive constellation designed to provide online servic

TED Fellow and economic policy researcher Huiyi Lin is cocreator of "The Poverty Line," an art project examining poverty through the lens of food. By photographing the daily food choices of people living at the poverty line in 38 countries and territories around the world, Lin shines a light on the problem of poverty in a way no policy report ever could.

The move to assume broad powers over the Providence school system illustrates both the attraction and challenges of high-profile approaches to troubled or low-performing schools and districts.

With the plan's approval, North Dakota's educators will experience some of the nation's most dramatic changes under the Every Student Succeeds Act this fall.

Attendance and login records show that Ohio's Electronic Classroom of Tomorrow was paid for 9,000 students more than it should have been, according to a state audit.